CDNAS encoding somatotropin, expression vectors and hosts

ABSTRACT

PCT No. PCT/US88/00328 Sec. 371 Date Aug. 18, 1989 Sec. 102(e) Date Aug. 18, 1989 PCT Filed Feb. 9, 1988 PCT Pub. No. WO88/06186 PCT Pub. Date Aug. 25, 1988.This invention discloses novel species of cDNAs encoding somatotropins, novel dual replicon vectors, hosts for such vectors and hosts for expressing the somatotropins encoded by such cDNAs.

This application is the U.S. national phase of international applicationPCT/US88/00328, filed Feb. 9, 1988 which is a continuation-in-part ofU.S. patent application Ser. No. 07/016,294, filed Feb. 19, 1987, nowabandoned.

FIELD OF THE INVENTION

This invention relates to the expression of heterologous polypeptides.More specifically, the invention discloses novel cDNAs encodingsomatotropins which are expressed at high levels in E. coli. A genericexpression system comprising a mutated E. coli host cell and anexpression vector having a dual replicon arrangement also is disclosedherein.

INFORMATION DISCLOSURE

Expression of somatotropins from a variety of animals by transformedmicroorganisms is known (Goeddel, D. V. et al., "Direct Expression inEscherichia coli of a DNA sequence coding for human growth hormone",Nature, 281:544-548 (1979); and Seeburg, P. H. et al., "EfficientBacterial Expression of Bovine and Porcine Growth Hormones", DNA,2:37-45 (1983)).

Naturally occurring bovine somatotropin (BSt) is a mixture ofheterogeneous proteins, the amino acid sequences of which are known(Paladini, A. C., et al., Molecular Biology of Growth Hormone, CRCReviews in Biochem., 15(1):25-56 (1983)). The naturally occurringmixtures have been purified from pituitary glands of cattle. Thecommercial potential for the use of BSt for promoting growth andlactation is well recognized and documented by biological studies onboth dairy and feed cattle (Eppard, P. J. and Bauman, D. E., The Effectof Long-Term Administration of Growth Hormone on Performance ofLactating Dairy Cows; and Bauman, D. E., Effect of Growth Hormone onGrowth Rates and Mammary Development of Ruminants, Proc. 1984 CornellNutrition Conference for Feed Manufacturers, pp. 5-17, published byCornell University, Ithaca, N.Y.).

Recobinant bovine somatotropin (rBSt) can be produced in transformedmicroorganisms using a variety of recombinant genetic plasmids (EuropeanPatent Application 47 600; United Kingdom Patent Application, GB2073245A; and Schoner, B. E. et al., Role of mRNA TranslationalEfficiency in Bovine Growth Hormone Expression in Escherichia coli,Proc. Natl. Acad. Sci. USA, 81:5403-5407 (1984)).

Analogs of BSt are also known (European Patent Application 103 395; andSchoner, B. E., et al., supra). Unlike the present invention, theseanalogs of BSt relate to the insertion or deletion of bases at the 5'end of the BSt gene thereby creating a protein different from thenaturally-occurring amino acid sequences. Modifications to rBSt cDNAs tomaximize preferred codons include changing the first two native alaninecodons of GCC to GCT (European Patent Application 111,814). However, EP111,814 teaches that preferred codon substitution and reduction ofsecondary structure is critical towards optimizing expression. Theinstant invention demonstrates that many of these known changes are notnecessary to achieve high levels of expression when the cDNAs arecombined with a runaway-replicon-type plasmid.

Methods for culturing and fermenting transformed microorganismsexpressing BSt are also known and fully described in the above-citedliterature.

Purification of biologically active rBSt from transformed cells has alsobeen described previously (U.S. Pat. Nos. 4,511,502; 4,511,503;4,512,922; 4,518,526; European Patent Application 131 843; and, Schoner,R. G., et al., "Isolation and Purification of Protein Granules from E.coli Cells Overproducing BSt", Bio-Tech., 3:151-154 (1985)).

The instant invention discloses cDNAs encoding somatotropins and analogsof somatotropins that are expressed at high levels when compared to thenative somatotropin cDNA. It is known that native cDNAs of porcine andbovine somatotropin are not expressed at commercially acceptable levelsin most E. coli systems and that changes in the cDNA are required forsuch expression. Attempts to increase the percentage of preferred codonsin the cDNA have had little affect and workers have had to resort tosubstantial modifications of the cDNA to reach acceptably high levels ofexpression. Seeburg, P. H. et al., DNA, 2:37-45 (1983) increasedexpression by eliminating the strong secondary structure resident afterthe condon for amino acid residue 11 in the native cDNA of bovinesomatotropin (see also European patent application 75444). Theypostulated that eliminating the secondary stem loop structures wouldenhance expression; this work directs the reader's attention to regionsof hydrogen bonding in excess of -12K calories/mole.

The instant invention teaches that high levels of expression need notinvolve substantial base sequence alterations. Rather, by making minorbasepair changes in the first four codons of the rBSt cDNA, one caneffect a substantial increase in expression in nonrunaway plasmids. Whencoupled with runaway plasmids, the cDNAs of the instant invention areexpressed at even higher, commercially acceptable levels. The use ofrunaway plasmids to express rBSt is known (European patent applications159,123 and 111,814). Neither application discloses the exact DNAsequences disclosed herein for the amino terminus of rBSt.

European patent application 103,395 discloses that expression of rBSt inE. coli will be at least 100 times higher than for the native cDNA ofBSt if the first three to nine triplet codons are deleted. EPapplication 103,395 also indirectly suggests the possibility of usingpoint mutations in the early condons to eliminate secondary structuralinterference with the Shine-Dalgarno region, but concluded that suchchanges could not be achieved with significant affect without alteringthe primary amino acid sequence of that part of the BSt protein.

SUMMARY OF THE INVENTION

This invention relates to expression plasmids, preferably containing arunaway type replicon, useful for transforming E. coli host cellspermitting the host cells to produce a somatotropin-like protein whereinthe plasmid contains the cDNA encoding a somatotropin selected from thegroup consisting of bovine, porcine and ovine somatotropin, and analogsthereof, wherein the first four codons encoding the somatotropin areselected from the group consisting of: ##STR1##

The preferred runaway-type replicons are those derived from R1 mutationscommercially available from A/S Alfred Benzon of Copenhagen, Denmark.Specifically those replicons found in Benzon's pBEU# series, i.e.,pBEU-50 or pBEU-17 which are identical runaway replicons. Most preferredare those expression plasmids in which the runaway replicon is placed incombination with the original replicon from pBR322. These chimericplasmids produce exceptionally high level expression of somatotropins inE. coli. The preferred E. coli host cells are those carrying mutationsof either the rpoH or the hflB gene (see below). Most preferred arethose host cells carrying the rpoH112 or hflB29 alleles (see below).

The above-described expression plasmids having a dual repliconarrangement are of general use for the expression of heterologous genesand are particularly advantageous when combined with the preferred E.coli host cells having mutations in either the rpoH or hflB gene,preferably, the rpoH112 and hflB29 alleles. In addition to theexpression of somatotropins, this combination of host and vector isuseful for the expression of biologically important proteins such aschymosin, interferon or interferon-like proteins, interleukins, insulin,viral proteins, urokinase, colony stimulating factor, tumor necrosisfactor, protein C and the like.

DEFINITIONS

The term "expression unit" means a DNA sequence containing a completeunit of gene expression and regulation, including structural genes,regulator genes and control elements which are necessary fortranscription, translation and for recognition by regulator geneproducts.

The term "heterologous", when referring to a gene, indicates that thegene has been inserted into a host cell either by way of a stableplasmid or through integration into the genome, and, when referring to aprotein, indicates that the protein is the product of a heterologousgene. Heterologous proteins are proteins that are normally not producedby a host cell or are normally produced in limited quantities.

"Native" refers to the sequence of amino acids or nucleic acidsoccurring from the natural source. A "native protein" is a proteinhaving a primary structure identical with the naturally occurringprotein. For an oligonucleotide or codon, the native nucleic acidsequence is identical to that occurring in nature, e.g., the basepairsequence of a cDNA synthesized from a naturally occurring mRNA would bethe native sequence. For recombinant genes or proteins, the nativesequence optionally includes the presence of the initiation codon ATGencoding methionine or a methionine residue present at the aminoterminus.

"BSt" refers to bovine somatotropin which includes a heterogenousmixture of proteins having ala-phe-pro-ala-met or phe-pro-ala-met at theamino terminus. For purposes of this application, the first codon orresidue will be considered to be alanine for numbering purposes. Relatedterms include bovine growth hormone, BGH, recombinant bovinesomatotropin, or rBSt. Recombinant BSt may optionally have an additionalmethionine residue at its amino terminus.

"Replicons" refer to DNA sequences that control the replication ofrecombinant DNA cloning and expression plasmids.

"Runaway replicons" contain sequences which either lack, or can beinduced to lose, copy number control. Such loss results in uncontrolledreplication and an increase in the copy number of the DNA molecule intowhich the runaway replicon has been incorporated. Related terms includerunaway-type plasmids or runaway plasmids which are plasmids containingrunaway replicons.

"Somatotropin(s)" refers to mammalian, fish and avian growth hormones.Unless restricted by the term "native", the term "somatotropin(s)"includes analogs of these proteins wherein there is sufficient aminoacid sequence identity to permit growth hormone activity to bemaintained. Such analogs include recombinantly produced somatotropinshaving the same amino acid sequences as the heterogeneous species foundin purified preparations from pituitary glands and analogs artificiallycreated by modification of somatotropin encoding DNAs such as aredescribed in European patent applications 103,395 and 75,444. Inaddition, in the primary sequence of rBSt, any L-amino acid may besubstituted for a D-isomer thereof and various amino acids may beinterchanged without affecting the activity of the molecule itself.Thus, for example, (1) alanine, leucine, isoleucine, valine and prolineare interchangeable, (2) phenylalanine and tryptophan areinterchangeable, (3) serine, threonine and tyrosine are interchangeable,(4) asparagine and glutamine are interchangeable, (5) lysine, arginine,histidine and ornithine are interchangeable, and (6) aspartic acid andglutamic acid are interchangeable.

"Transformed microorganism" refers to a prokaryotic or eukaryotic singlecelled organism containing or hosting a plasmid artificially insertedinto the cell using techniques well known in molecular genetics.

DETAILED DESCRIPTION

This invention provides methods for preparing mutant cDNA sequences thatresult in high level expression of eukaryotic growth hormone genes inrecombinant microorganisms. In particular, the construction of severalcDNAs useful for obtaining high level synthesis in E. coli of maturebovine and porcine growth hormones in different vectors is illustrated.For comparison, amounts of products expressed are presented for plasmidscontaining BSt cDNAs with and without nucleotide changes. Table 1 setsforth the coding sequence of mature BSt cDNA and Table 2 sets forth themutations introduced into the 5' region of the BSt cDNA to enhanceexpression when compared to the native cDNA. The oligonucleotides ofTable 2 were synthesized chemically according to the methods set forthbelow.

A. General Methods

Generally, the definitions of nomenclature and descriptions of generallaboratory procedures used for this invention can be found in Maniatis,T. et al., Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982). The manual is hereinafterreferred to as "Maniatis" and is incorporated herein by reference.

All E. coli strains are grown on Luria broth (LB), LB with glucose,Difco's Antibiotic Medium #2, or M9 medium supplemented with glucose andacid-hydrolyzed casein amino acids. Strains resistant to antibiotics aremaintained at the drug concentrations described in Maniatis.

Transformations are performed according to the method described byMorrison, D. A., J. Bacteriol., 132:349-351 (1977), or by Clark-Curtiss,J. E. and Curtiss, R., Meth. Enzymol., 101:347-362 (1983).

All enzymes are used according to the manufacturer's instructions.Restriction fragments are separated by either agarose or polyacrylamidegel electrophoresis and isolated by electroelution (Maniatis) or byadsorption onto glass powder (Vogelstein, B. and Gillespie, D., Proc.Natl. Acad. Sci. USA, 76:615-619 (1979)).

Large scale and rapid plasmid isolations are done as described inManiatis.

Protein concentration is determined using the BioRad protein assay kit,based on Coomassie Blue binding.

SDS polyacrylamide gel electrophoresis for protein analysis is performedas described in Morse, L., et al., J. Virol., 26:389-410 (1978), andLaemmli, U. K., Nature (London) 226:680-685 (1970).

Western immunoblotting analysis was performed as described in Towbin,H., et al., Proc. Natl. Acad. Sci. USA, 76:4350-4354 (1979).

Colony hybridization is carried out as generally described in Grunstein,M. et al., Proc. Natl Acad. Sci. USA, 72:3961-5 (1975). Filters are thenthoroughly air dried and baked in vacuo for 2 hours at 80° C.

Hybridization conditions for oligonucleotide probes are as previouslydescribed by Goeddel, D. V. et al., Nature, 290:20-26 (1981). Afterhybridization, the probe containing solution is removed and saved andthe filters are washed in 0.1% SDS, 5×SSC. Filters are air dried,mounted, and autoradiographed using Kodak X-OMAT AR film and DupontCronex Lightning Plus intensifying screens at -70° C.

For sequencing plasmids, mini-lysates of plasmid DNA are preparedaccording to the method of Holmes, D. S. et al., Anal. Biochem., 114:193(1981), or by the alkaline-lysis procedure described in Maniatis.Dideoxy sequencing is carried out according to Sanger, F. et al., J.Mol. Biol., 143:161-178 (1977) using double-stranded plasmids. Thedideoxy-containing reactions are prepared for electrophoresing byheating to 90° C. for 2 min, and quenching on ice. 2-3 μl per lane isloaded on an 8% denaturing polyacrylamide gel prepared and run accordingto Sanger and Coulson, FEBS Lett., 87:107-110 (1978).

Nucleotide sizes are given in either kilobases (kb) or basepairs (bp).

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester method(Beaucage, S. L. and Caruthers, M. H., Tetrahedron Letts.,22(20):1859-1862 (1981)) using an automated synthesizer, as described inNeedham-VanDevanter, D. R. et al., Nucleic Acids Res., 12:6159-6168(1984). After the oligonucleotides are purified, they are desalted on aWaters Sep-Pak C18 column.

The sequence of the synthetic oligonucleotides is verified using thechemical degradation method of Maxam, A. M. and Gilbert, W., Methods inEnzymology, 65:499-560 (1980). Alternatively, the sequence can beconfirmed after assembly of the oligonucleotide fragments into plasmidsusing the method of Maxam and Gilbert, supra, or the chain terminationmethod for sequencing double-stranded templates of Wallace, R. B. etal., Gene, 16:21-26 (1981).

To assemble the oligonucleotides, the 5'-OH ends of the oligonucleotidesto be ligated within the linker are phosphorylated with T4polynucleotide kinase in the presence of 2 μCi of γ-³² P-ATP per μg ofoligonucleotide, followed by a chase with an excess of unlabelled ATP.Annealing is performed in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, byheating to 90° C. for 10 min, followed by slow cooling to roomtemperature over a period of 2-4 h. After incubation at 15° C. for 1 hand addition of ATP to 0.5 mM, DTT to 20 mM, and 400 U T4 DNA ligase,the reaction mixture is incubated at 15° C. overnight. The correctlyassembled linker is purified by 10% polyacrylamide gel electrophoresisusing the labelled HaeIII digested φX174 DNA as a standard, followed byelectroelution (Maniatis) and precipitation with ethanol.

B. Expression in Prokaryotes

To obtain high level expression of a cloned gene in a prokaryoticsystem, it is essential to construct expression vectors which contain,at the minimum, a strong promoter to direct mRNA transcription, and aribosome binding site for translational initiation. Since theaccumulation of large amounts of a gene product often inhibits cellgrowth and sometimes causes cell death, the promoter chosen to directthe synthesis of the product should be regulated in such a way that cellgrowth can be allowed to reach high densities before the induction ofthe promoter. Examples of regulatory regions suitable for this purposeare the promoter and operator region of E. coli tryptophan biosyntheticpathway and the leftward promoter of phage lambda (P_(L)). The trppromoter is repressed in the presence of tryptophan and can be inducedby tryptophan starvation or by the addition of the inducer indoleacrylic acid (Yanofsky, C., et al., J. Bacteriol., 158:1018-1024(1984)). Promoter P_(L) is controlled by the repressor cI. With atemperature-sensitive mutation in the cI gene, e.g., cI857, P_(L) can beinduced at temperatures above 38° C. (Herskowitz, I. and Hagen, D.,Annu. Rev. Genet., 14:399-445 (1980)). Most preferred are expressionvectors having restriction enzyme sites at an appropriate distance fromthe Shine-Dalgarno sequence in order to insert genes to be expressed.

To synthesize a protein encoded by a eukaryotic gene from its cDNAsequence in E. coli intracellularly, it is expedient to remove the 5'untranslated region and the sequence coding for the signal peptide andto supply an initiation codon for translation initiation of the sequencecoding for the protein. It may also be necessary to replace some of thecoding sequence for the protein with chemically synthesizedoligonucleotides to maximize translation efficiency. The instantinvention specifically describes a truncated BSt gene having the 5'untranslated region, the pre-sequence, and a 66 bp coding sequence forthe beginning of mature BSt deleted. This truncated BSt sequence iscloned into our expression vector pTrpl to yield plasmid pTrp-BStmlb. Tocomplete the truncated BSt sequence for expression, oligonucleotides areinserted into pTrp-BStmlb. The oligonucleotides can be designed toincorporate changes leading to optimal expression with or withoutalteration in amino acid residues.

A preferred embodiment of this invention utilizes a high-copy numberplasmid or a runaway-type origin of replication. All plasmids are knownto contain a DNA sequence from which replication of the plasmid occurs.These sequences are diverse in nature and are generally referred to asorigins of replication (ori) or replicons. In some cases the ori canfunction by using E. coli host proteins for replication, while othersrequire additional protein factors encoded by the plasmid.

The number of copies of a plasmid per cell (i.e., copy number) can beaffected by changes in the replication ori and in the regulation of theplasmid encoded genes. Vector mutations have been isolated which causeincreased plasmid replication either constitutively or under controlledconditions. Examples of constitutively high-copy-number plasmids arepUC19 (Yanisch-Perron, C. et al., Gene, 33:103-119 (1985), availablefrom Bethesda Research Labs, Gaithersburg, MD, USA); and pHC314 (Boros,I. et al., Gene, 30:257-260 (1984)). The increase in plasmid replicationunder controlled conditions is referred to as plasmid "runaway". Thecontrol mechanism is usually a shift in culture temperature. Thepreferred "runaway" vector system that is used here was derived from theR1 plasmid. Temperature sensitive mutations of vector derivatives of R1were isolated in a two-step selection (Uhlin and Nordstrom, Mol. Gen.Genet., 165:167-179 (1978); and Uhlin et al., Gene, 6:91-106, (1979)).Upon heat induction these vectors can comprise as much as 75% of thetotal DNA in the cells.

The replication region of the R1 plasmid is clustered on about 2.5 kb ofDNA sequence (Ryder et al., Gene, 17:299-310 (1982)). This regioncontains at least three genes and the origin of replication (Light andMolin, EMBO J., 2:93-98 (1983); Light et al., Mol. Gen. Genet.,198:503-508 (1985)). The genes are copB, copA, and repA. The productionof the repA protein acts to initiate replication at the ori. The numberof initiation events is proportional to the amount of the repA proteinproduced. The function of the copA and copB gene products is to regulatethe amount of repA protein made. The copB product is a repressormolecule which binds to a repA specific promoter (Riise and Molin,Plasmid, 15:163-171 (1986); Givskov et al., Gene, 57:203-211 (1987)).The repA gene is also expressed from the upstream copB promoter. ThecopA gene has its own promoter transcribed in the opposite direction tocopB and repA and produces an antisense RNA which hybridizes to the repAmRNA and decreases the efficiency of translational initiation of therepA mRNA.

The Benzon vectors used herein, such as pBEU-50 and pBEU-17, werederived from the original two-step temperature controlled runaway vectorderived from R1 (Uhlin and Nordstrom, supra; Uhlin and Clark, In:Molecular Biology, Pathogenicity, and Ecology of Bacterial Plasmids,eds. S. B. Levy, R. C. Clowes and E. L. Koenig, Plenum Press, p. 670(1981)). These vectors contain two promoter mutations. The first is amutation in the copA promoter which reduces the amount of antisense copARNA and the second is a mutation in the copB promoter which increasestranscriptional initiation at elevated temperatures (Givskov et al.,supra). E. coli cells containing the pBEU-50 vector are grown attemperatures of 30° C. or lower. When the cells are shifted totemperatures above 34° C. the plasmid copy number increases and this inturn amplifies the expression of genes which have been cloned into thevector (Uhlin et al., supra).

A chimeric vector composed of the pBR322 replicon and the runawayreplicon of pBEU-17 expresses the modified somatotropins of the instantinvention at particularly high levels. When placed in combination withtwo E. coli strains exhibiting low levels of protease activity,expression levels become extremely high.

Several genes in E. coli are known to affect the expression of proteasesin the cell. These genes include lon, hflA, hflB, and rpoH (Cell,41:587-595 (1985); Proc. Natl. Acad. USA 81:6647-6651 (1984); Proc.Natl. Acad. Sci. USA 81:6779-6783 (1984); Cell 31:565-567 (1982);Genetics 77:435-448 (1974)). Strains containing either a rpoH or hflBmutation have demonstrated enhanced expression of rBSt, as modifiedherein, and particularly in combination with a runaway plasmid.Preferred alleles are rpoH112 and hflB29. Other low protease strains areunable to enhance expression of rBSt.

Techniques for isolation and purification of mammalian somatotropinsfrom recombinant microorganisms are well known in the art (see, e.g.,U.S. Pat. Nos. 4,511,502, 4,512,922, 4,518,526; European PatentApplication 131,843; and Schoner, R. G., et al., supra). In brief, theprocess involves lysing the cells, selective centrifugation, reshufflingof any non-native disulfide bonds to the native configuration and columnchromatography.

Conventions used to represent plasmids and fragments in Charts 1-14 areas follows: the single line figures on the charts represent bothcircular and linear double-stranded DNA with translation initiation ortranscription occurring in the direction of the arrow where indicatedbelow a promoter or structural gene. Asterisks (*) represent thebridging of nucleotides to complete the circular form of the plasmids.Fragments do not have asterisks because they are linear pieces ofdouble-stranded DNA. Endonuclease restriction sites are indicated abovethe line. Gene markers are indicated below the line. The relativespacings between markers do not indicate actual distances but are onlymeant to indicate their relative positions on the illustrated DNAsequence.

EXAMPLE 1 RBSt Expression in Non-runaway Vectors

A) The Expression Vector pTrpl

A prokaryotic expression vector for expressing heterologous proteins inmicroorganisms preferably has a strong regulatable promoter to mediatetranscription and a strong ribosome binding site for translationinitiation. For expression of mature BSt in E. coli, we have constructedthe expression vector pTrpl which is derived from pSK4 (Kaytes, P. S.,et al., J. Biotech., 4:205-218 (1986)). pTrpl contains the promoter andoperator sequence of the E. coli tryptophan operon, the Shine-Dalgarnosequence of the trpL gene, the replication origin from pBR322 and a genefor ampicillin resistance. pTrpl has a unique ClaI site following thetrpL Shine-Dalgarno sequence and a unique KpnI/Asp718 site immediatelyafter the initiation codon, ATG. Insertion of a gene having aninitiation codon at the ClaI site of pTrpl allows expression of thatgene. Insertion of a gene at the KpnI/Asp718 site results in theexpression of that gene as a fusion with amino acid gln or gln and valdepending upon which site is used.

To make pTrpl, two complementary oligonucleotides are inserted intopSK4, as shown in Chart 1. Plasmid pSK4 contains the promoter/operatorregion of the trp operon and the trpL ribosome binding site cloned intopBR322 which also specifies resistance to ampicillin. The largeBamHI/ClaI fragment including the trp sequences is cut out from pSK4 andpurified by electroelution from agarose gels (fragment 1). Twocomplementary oligonucleotides are combined to form fragment 2. Fragment2 has a ClaI and a BamHI sticky end at the 5'- and 3'-end, respectively,and is ligated to fragment 1. Clones with the KpnI/Asp718 site andrestriction patterns characteristic of the trp promoter are chosen andare called pTrpl. The sequence downstream from the trp promoter in pTrplis verified by sequencing.

B) Construction of a Truncated BSt Gene

To delete the 5'-untranslated region, the pre-sequence, and the sequencecoding for the beginning of BSt, the 494 bp PvuII fragment 3 is isolatedfrom pLG23 (Chart 2). pLG23 was deposited in accordance with theBudapest Treaty on May 12, 1981 with the Northern Regional ResearchLaboratory in Peoria, Ill., USA and assigned Accession Number NRRLB12436. pLG23 contains the full length cDNA for BSt, including the 5'and 3' untranslated regions, cloned into the PstI site of pBR322. Thehost microorganism is E. coli HB101. The characteristics of this plasmidhave been fully described in European patent application 67026 publishedon Dec. 15, 1982 and in U.S. patent application, Ser. No. 269,187, filedJun. 1, 1981, both of which are incorporated herein by reference.Fragment 3 contains the cDNA sequence coding for amino acid residues 24to 188 of BSt (see Table 1). Fragment 3 is then ligated to pTrplpreviously digested with KpnI and treated with Klenow fragment to removethe sticky ends (fragment 4). Ligation of the BSt fragment to the vectorin the desired orientation produces a BSt cDNA sequence missing thecodons for the first 22 amino acid residues. The desired clone, selectedby the characteristic restriction patterns and confirmed by DNAsequencing, is named pTrp-BStml.

The BSt cDNA contains 2 PvuII sites, one at the codon for amino acidresidue 23 and one at residue 188. For ease of manipulation of thetruncated BSt sequence, the second PvuII site is removed in pTrp-BStml.To remove this PvuII without changing the amino acid residues, the 56 bpregion between MstII and BamHI in pTrp-BStml is replaced witholigonucleotides. In chart 3, the large BamHI/MstII fragment 5 isisolated from pTrp-BStml and ligated to 4 oligonucleotides assembled andisolated to produce fragment 6. The resulting plasmid, calledpTrp-BStmlb, is selected by characteristic restriction patterns andverified by DNA sequencing. In pTrp-BStmlb, the BSt sequence istruncated as in pTrp-BStml, i.e., missing the codons for the first 22amino acids. The sequence at the 3'-end after the MstII site is modifiedto remove the PvuII site and to introduce a HindIII site, withoutchanging the amino acid sequence. Two stop codons followed by a BamHIsite are introduced immediately at the end of the gene.

C) Construction of plasmids for BSt expression

To obtain high level expression of mature BSt, four oligonucleotides areused to replace the small ClaI to PvuII region in Trp-BStmlb to supplythe missing BSt sequence. Due to the presence of another PvuII site inthe vector, it is simpler to separate the vector into two portions. Asshown in Chart 4, the 4.3 kb MstII/ClaI fragment 7 is isolated frompTrp-BStmlb. This fragment contains the trp promoter/operator, trpLShine-Dalgarno sequence, pBR322 replication origin, ampicillinresistance determinant, and the 3'-end of the BSt gene. The 450 bpPvuII/MstII fragment 8 containing the sequence coding for BSt amino acidresidues 24 to 176 is also isolated from pTrp-Bstmlb. Fragments 7 and 8are ligated to fragment 9, the block of 4 oligonucleotides alreadyassembled and purified. The resulting plasmid, selected by colonyhybridization using one of the 4 oligonucletides as a probe andconfirmed by DNA sequencing, is called pTrp-BStm4. In pTrp-BStm4, theonly change made at the beginning of the BSt gene is changing the codonfor alanine at position 4 from GCC to GCT.

To construct plasmids for expressing BSt with other changes at thebeginning of the gene, other oligonucleotides assembled as 4 in a blockare used to ligate to fragments 7 and 8. In Table 2, the oligonucleotideblocks used and the resulting plasmids are listed. The changes made areindicated by the asterisked nucleotide and listed below. The relativeability of these constructs to express BSt in E. coli K12 is as follows:

    ______________________________________                                                               BSt Expression                                         Plasmid                % of Total Protein                                     ______________________________________                                        pTrp--BSt102, no changes at the beginning                                                            <0.01%                                                 of BSt.                                                                       pTrp--BStm4, ala 4 changed from GCC to                                                               1%                                                     GCT                                                                           pTrp--BStm5, ala 1 and ala 4 changed from                                                            1%                                                     GCC to GCT.                                                                   pMBSt4, ala 1 changed from GCC to thr                                                                1%                                                     (ACC).                                                                        pMBSt12, ala 4 changed from GCC to val                                                               1%                                                     (GTC).                                                                        pTrp--BSt[phe], ala 1 is changed to                                                                  2-5%                                                   phe (TTC).                                                                    pTrp--BSt[lys], ala 1 is changed to lys                                                              2-5%                                                   (AAA).                                                                        pTrp--BStm3, ala 1 is deleted.                                                                       15                                                     ______________________________________                                    

EXAMPLE 2 Inductions With The cDNA Derived Genes 102, m4 and m5 in aRunaway Vector

A) Construction of Runaway Vectors p50-102, p50-BStm4 and p50-BStm5

The enhanced expression mutations of the BSt gene constructed accordingto Example 1 can also be used in plasmids having a runaway replicon. Thevector source for the runaway replicon is designated pBEU-50 and iscommerically available from A/S Alfred Benzon of DK-1700 Copenhagen,Denmark. This vector is about 10 kb in size and carries the temperaturesensitive copB promoter and a down-promoter for copA. The vector alsocarries the resistance genes to tetracycline (tet) and ampicillin (amp).

As shown in Chart 5, plasmid DNA of pBEU-50 was digested with EcoRI andBamHI endonucleases, which each cut the vector DNA once. The digestionproduces a 0.375 kb and a 10 kb fragment. The 10 kb fragment wasisolated from an agarose gel by electroeluting (fragment 10).

The EcoRI/BamHI fragments containing the rBSt gene were isolated frompTrp-BSt102, pTrp-BStm4 and pTrp-BStm5 by digesting with EcoRI andBamHI. This digestion generates a 3.9 kb and a 0.872 kb fragment. The0.872 kb fragment (fragment 11) was isolated from an agarose gel asdescribed above. Fragment 11 contains approximately 240 bp encoding thetryptophan promoter and tryptophan leader ribosomal binding site. Theremainder of the fragment encodes the rBSt cDNA gene.

The three rBSt cDNA fragments (exemplified by fragment 11 in Chart 5)were each cloned by ligating the fragments with fragment 10 isolatedfrom pBEU-50. The plasmids so produced were transformed into competentE. coli cells (Maniatis). Replacement of the original EcoRI/BamHIfragment with the rBSt fragment functionally destroys the tetracyclineresistance gene. Colonies from the transformation were screened fortetracycline sensitivity and DNA from selected colonies was analyzed byrestriction digestions to confirm the construction. The resultantvectors were referred to as p50-102, p50-BStm4 and p50-BStm5. p50-102containing no modifications in the beginning of BSt was constructed tocompare with p50-BStm4 and p50-BStm5 where the BSt was modified at thecodons for ala4 (p50-BStm4) and alal and ala4 (p50-BStm5). By using thesame method, the other novel cDNAs from Example 1 can be used toconstruct similar plasmids designated p50-MBSt4, p50-MBSt12, p50-BSt[phe], p50-BSt[lys], and p50-BStm3.

B) Construction of Dual Replicon Runaway Vectors

The following example provides details on the construction of a novelcombination of replicons useful for the expression of the somatotropinsdescribed herein. This class of chimeric vectors are designated pURA.The first step in the construction involves the isolation of the runawayreplicon and associated genes from the Benzon family of plasmids (seeChart 6). A 2.4 kb AhaII-NdeI fragment (fragment 12) was isolated from apBEU-50-related Benzon vector called pBEU-17 (13.8 kb) which iscommercially available from A/S Alfred Benzon. Fragment 12 (3.6 kb)contains the entire replication region of the vector and was cloned intothe NarI and NdeI sites of pBR322. The cloning replaces about 2 kb ofpBR322 with the 2.4 kb from pBEU-17. The AhaII and NarI restrictionsites share complementary ends and a NarI site is regenerated. The NdeIsite is also regenerated. The vector contains a double origin ofreplication, one from pBEU-17 and one from the pBR322 vector. Theresultant vector is designated pURA (5 kb). This vector also containsthe unique restriction sites for EcoRI and BamHI which were present inthe original pBR322 plasmid.

To express a somatotropin using the chimeric plasmids of the pURAseries, for example, the BSt encoded by pTrp-BStm4 from Example 1 (them4 gene), the expression vector pURA-m4 was constructed from a tripleligation (Chart 7). Ligated were: (1) an isolated EcoRIBamHI fragment(875 bp) from p50-BStm4 (Chart 5 ) which contains the trp promoter, thetrp leader ribosomal binding site, and the m4 gene (fragment 13); (2) atranscription terminator for the E. coli genes rpoBC that was isolatedas a 350 bp BamHI fragment (fragment 14) from the plasmid vector pVV202T(obtained from Dr. Charles Yanofsky, Stanford Medical School); and, (3)a pURA plasmid vector fragment (4.6 kb, fragment 15) that was isolatedafter pURA was digested with EcoRI and BamHI. Fragments 13-15 weremixed, ligated and transformed into competent E. coli cells. Vector DNAfrom the transformed clones was analyzed, and a vector that had theterminator in the same orientation with respect to the transcription ofthe m4 gene as it is to the rpoBC genes in the E. coli chromosome wasdesignated pURA-m4 (5.8 kb). A schematic of pURA-m4 is presented inChart 7.

It should be noted that the particular transcription terminator chosenis not the only terminator useful in this invention. Any of a number ofefficient rho independent transcription terminators could besubstituted. For a review of these terminators see Cell, 32:1029-1032(1983) and Ann. Rev. Genet., 13:319-53 (1979).

In a like fashion the BSt genes from pTrp-BStm5, p50-MBSt4, p50-MBSt12,p50-BSt[phe], p50-BSt[lys], and p50-BStm3 can be inserted into pURA toobtain pURA-m5 pURA-MBSt4, pURA-MBSt12, pURA-BSt[phe], pURA-BSt[lys],and pURA-BStm3.

C) Construction of an E. coli Host for rBSt Expression

The E. coli progenitor strain was obtained from the American TypeCulture Collection and is designated ATCC e23716. This strain islysogenic for the bacteriophage lambda and also harbors the F plasmid(Bachmann, B. J., Bact. Rev. 36:525-557 (1972)).

The lambda lysogen was removed from the strain. This was accomplished bythe Plvir bacteriophage transduction technique as described by Miller,J. H. in Experiments in Molecular Genetics, Cold Spring HarborLaboratory (1972). The Plvir bacteriophage is available as a part of the"Experiments with Gene Fusion Strain Kit" from the Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y.

Bacteriophage Plvir was grown up on E. coli strain CGSC 6180 which isavailable from the Coli Genetic Stock Center, c/o Dr. Barbara Bachmann,Yale University, New Haven, Ct. 06510. This strain is not lysogenic forthe bacteriophage lambda, and contains a Tn10 element inserted into thenadA gene (which encodes the A protein of the quinolinate synthetase)and is located adjacent to the integration site of the bacteriophagelambda. The Tn10 element destroys the nadA gene and causes the cell tobe dependent on an exogenous source of nicotinamide. The Tn10 elementcarries a gene which codes for resistance to tetracycline. The Plvirlysate from CGSC 6180 (nadA::Tn10) was used to transduce the nadA::Tn10allele into the ATCC e23716 by selecting for resistance to tetracycline.A number of colonies from the selection were tested for recombinationalloss of the adjacent lambda phage by sensitivity to bacteriophage T4rII(Benzer, S., Proc. Natl. Acad. Sci. USA, 47:403-408 (1961)). ThenadA::Tn10 allele was removed by growing Plvir on W3110 strain(available from the Coli Genetic Stock Center, New Haven, Ct. which hasthe normal nadA gene and is not lysogenic for lambda. Selection for thenormal nadA allele was performed by plating the cells on a medium thatwas not supplemented with nicotinamide.

Next the F plasmid was removed by growing cells in the presence of 4μg/ml rifampicin for multiple generations. Cells that had lost the Fplasmid were identified by their inability to support the growth of theF plasmid-specific bacteriophages (Caro, L. G. and M. Schnos, Proc.Natl. Acad. Sci. USA, 56:126-131 (1966)). The resultant strain wasdesignated K12D.

C) Induction of the p50-102, p50-BStm4 and p50-BStm5 Vectors in the K12DStrain

The vectors were transformed into competent K12D cells by selecting forampicillin resistance. The three cultures were grown overnight in LBmedia containing 100 μg/ml ampicillin. The following day the cells weresubcultured 1/50 into the same media, and incubated at 27° C. using asterile flask in a New Brunswick shaking incubator set at about 275 rpm.The cultures were grown to an O.D._(550nm) of 0.2 to 0.3., and thenshifted to 38.5° C. to induce runaway replication.

Samples were taken after the inductions and were analyzed by SDS-PAGEgel analysis (Laemmli, supra). The gels were stained with Coomassie Blueand were scanned to determine the amount of visible rBSt.

There was no detectable rBSt for the inductions of p50-102. The culturescontaining either p50-BStm4 or p50-BStm5 produced between 13 to 18 areapercent BSt.

A comparison with HPLC was made between the amount of rBSt present in a20 O.D. sample of induced cells containing p50-102 and p50-BStm4. Thep50-102 showed no detectable rBSt (0.00 mg/ml), and the p50-BStm4 showed179.02 mg/ml.

These results show that the single base change in the codon for thealanine at the fourth position of BSt from GCC to GCT causes high levelrBSt production in the runaway plasmids.

EXAMPLE 3 Construction of E. coli Hosts with the rpoH112 and hflB29Alleles

The E. coli strain CAG671 was obtained from Dr. Carol Gross (Universityof Wisconsin, Madison, Wis.). This strain contains the rpoH112 alleleand has an adjacent insertion zhg: : Tn10 (Grossman, A. D., et al., J.Bact., 161:939-943 (1985)). The rpoH112 allele was introduced into theK12D strain by growing the bacteriophage Plvir on the CAG671 strain andusing the lysate to transduce the K12D strain to tetracycline resistance(encoded in the Tn10 located adjacent to the rpoH112 allele) and toco-transduce in the rpoH112 allele (Miller, J. H., supra). The resultantstrain was designated D112 and was deposited in accordance with theBudapest Treaty on Feb. 4, 1987, with the Agricultural Research ServiceCulture Collection, Northern Regional Research Center, 1815 NorthUniversity Street, Peoria, Ill. 61604, USA and assigned Accession NumberNRRL B-18168.

The E. coli strain x9393 was obtained from Dr. Ronald Sommerville(Purdue University, Indiana). This strain contains the hflB29 allele,and an adjacent Tn10 element (Banuett, F. et al., J. Mol. Biol.,187:213-224 (1986)). The hflB29 allele was introduced into the K12Dstrain by the same procedure that was described for rpoH. The resultantstrain was designated B29.

EXAMPLE 4 Expression of BST Using E. coli Hosts with the rpoH112 andhflB29 Alleles and Dual Replicon Plasmids

The vector pURA-m4 was transformed into competent cells of E. colistrains K12D, D112 (K12D with rpoH112) and B29 (K12D with hflB29).Cultures were grown in shaker flasks containing LB broth in a 27° C. airshaker. At the desired time the cultures were shifted to a 37° C. shakerto induce runaway vector replication and rBST expression. Samples weretaken prior to the heat shift and at various times thereafter. Thesamples were analyzed by SDS-PAGE and gel scanning. The results showthat rBSt reached 13.3% of visible protein in K12D, 32% in B29, and 41%in D112.

These results show that expression of rBSt is enhanced by differentcombinations of runaway plasmids when the disclosed modifications in thenative cDNA are made but that there is a particularly advantageouselevation in expression levels when the chimeric plasmid is placed in anE. coli strain containing a rpoH or hflB allele.

EXAMPLE 5 Spontaneous Induction of Runaway Plasmid Replication and rBStSynthesis with pURA-m4.

A) Relationship Between Spontaneous Induction of rBSt Synthesis andSpontaneous Induction of Plasmid Runaway Replication

Cultures of strain D112 containing the runaway replication vectorpURA-m4 and adapted for fermentation (called BST-1, see section C below)were inoculated into flasks of defined inorganic salts mediasupplemented with glucose and either yeast extract or casamino acids.From shake flasks incubated at 28° C. and shifted to 37° C. at an O.D.of 1 A₅₅₀, samples were obtained and subjected to SDS-PAGE (Laemmli,supra). Such samples shows appreciable rBSt synthesis, due to thethermal inducibility of plasmid runaway replication and rBSt expressioncharacteristic of pURA-m4. However, when samples obtained from companionflasks containing the same media and inoculated with the same culture,but incubated at 28° C. throughout, were subjected to SDS-PAGE analyses,appreciable rBSt synthesis was also observed. These observations shownthat strain BST-1 can spontaneously (i.e., non-thermally) induce rBStsynthesis.

Fermentations done in defined inorganic salts media supplemented withglucose and yeast extract confirmed the ability of pURA-m4 to undergospontaneous induction of rBSt synthesis. Furthermore, thesefermentations established that the spontaneous induction of rBStsynthesis occurred at the same time as spontaneous induction of plasmidrunaway replication occurred. In the absence of thermal induction,pURA-m4 spontaneously increased to substantially higher copy numbers.The data in Table 3 depict the relationship between the spontaneousinduction of plasmid copy number and spontaneous induction of rBStsynthesis. By approximately 15 hours post-inoculation, rBSt accumulationwas detected in samples subjected to reversed-phase HPLC analysis. Asimilar temporal increase in plasmid copy number (plasmid DNA content)was observed for pURA-m4 during fermentations conducted at 28° C.throughout.

B) Spontaneous Inducibility of Plasmid Runaway Replication in theAbsence of rBSt Synthesis

Plasmid pURA4 Δbgh_(E/H) was constructed from plasmid pURA-m4 by removalof an approximately 850 base pair fragment bounded by EcoRI and HindIIIrestrictions sites, thus deleting the gene encoding rBSt synthesis(Chart 13).

Plasmid pURA4 Δbgh_(E/H) was introduced into E. coli strain BST-1C, aplasmid-free derivative of strain BST-1 obtained following growth ondefined minimal medium at 42° C. and found cured of pURA-m4, byDNA-mediated transformation. The resulting strain was unable tosynthesize rBSt. E. coli host strain BST-1C was deposited in accordancewith the Budapest Treaty on Feb. 2, 1988, with the Agricultural ResearchService Culture Collection, Northern Regional Research Center, 1815North University Street, Peoria, Ill. 61604, U.S.A. and assignedAccession Number NRRL B-18303.

Data from fermentations that were conducted in defined inorganic saltsmedia supplemented with glucose and yeast extract shown the ability ofstrain BST-1C (pURA4 Δbgh_(E/H)) to undergo spontaneous induction ofrunaway plasmid replication. When compared with plasmid-containingpreparations obtained from strain BST-1, preparations from BST-1C (pURA4Δbgh_(E/H)) established that plasmids from both strains hadspontaneously increased to levels which were very similar by the ends oftheir respective fermentations (Table 4). Therefore, spontaneousinduction of runaway replication to a substantially higher plasmid copynumber was independent of host rBSt synthesis per se.

C) Fermentation Conditions for Spontaneous Induction

Set forth here is the preferred fermentation protocol for the E. colihosts transformed with pURA plasmids.

The strain construction used in these experiments was designated BST-1.BST-1 was derived by adapting the D112 strain transformed with pURA-M4to the fermentation seed medium set forth below. BST-1 can also beproduced by transforming strain BST-1C (above) with p-URA-m4 (above).

Two fermentation media, RB6 and RB7, were used in the experiments. Thecomposition of these media and methods of preparation are as follows:

A) Medium Preparation Procedures:

1) Medium RB6:

    ______________________________________                                        Ingredient      Concentration                                                                             Amount                                            ______________________________________                                        Na(NH.sub.4)HPO.sub.4.H.sub.2 O                                                               11      g/l     99     g                                      K.sub.2 HPO.sub.4                                                                             2.6     g/l     24     g                                      Citric Acid.H.sub.2 O                                                                         2.1     g/l     19     g                                      Yeast Extract   1.0     g/l     9.0    g                                      (NH.sub.4).sub.2 SO.sub.4                                                                     0.66    g/l     5.9    g                                      MgSO.sub.4      0.25    g/l     2.2    g                                      SAG 4130        0.75    ml/l    6.8    ml                                     ______________________________________                                    

Q.S. the above ingredients to 8.3 l with R.O. water in a 16 l fermentor,sterilize at 121° C. for 20 minutes and then cool. Prior to inoculatingthe fermentor, aseptically add 675 ml of a sterile 500 g/l glucosesolution, 9 ml of sterile micronutrients and 9 ml of a sterile 25 g/lampicillin solution to the fermentor (See "Fermentation Additions"section below).

2) Medium RB7:

    ______________________________________                                        Ingredient      Concentration                                                                             Amount                                            ______________________________________                                        Na(NH.sub.4)HPO.sub.4.H.sub.2 O                                                               11      g/l     99     g                                      K.sub.2 HPO.sub.4                                                                             2.6     g/l     24     g                                      Citric Acid.H.sub.2 O                                                                         2.1     g/l     19     g                                      Yeast Extract   1.0     g/l     9.0    g                                      D,L-Methionine  0.75    g/l     6.7    g                                      (food grade)                                                                  (NH.sub.4).sub.2 SO.sub.4                                                                     0.66    g/l     5.9    g                                      MgSO.sub.4      0.25    g/l     2.2    g                                      SAG 4130        0.75    ml/l    6.8    ml                                     ______________________________________                                    

The remainder of the procedure is the same as that given for RB6.

3) Fermentation Additions:

a) Micronutrient Solution (200 ml):

    ______________________________________                                        Ingredient     Amount (grams)                                                 ______________________________________                                        FeSO.sub.4.H.sub.2 O                                                                         5.22                                                           Citric acid.H.sub.2 O                                                                        5.04                                                           ______________________________________                                    

Add 8 ml of trace components* to the above ingredients, q.s. to 200 ml,and sterilize by passage through a 0.22 micron filter.

(*) The trace component solution is prepared as follows:

    ______________________________________                                        Ingredient       Amount (mg)                                                  ______________________________________                                        Citric acid.H.sub.2 O                                                                          7000                                                         H.sub.3 BO.sub.4 247.4                                                        MnCl.sub.2.4H.sub.2 O                                                                          158.0                                                        CoCl.sub.2.6H.sub.2 O                                                                          71.4                                                         (NH.sub.4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O                                                  37.1                                                         ZnSO.sub.4.7H.sub.2 O                                                                          28.8                                                         CuSO.sub.4       15.9                                                         ______________________________________                                    

Q.S. to 100 ml with distilled water.

b) 500 g/liter glucose addition (675 ml):

Q.S. 338 g of glucose in a 1/liter shake flask to 675 ml with R.O.water, add sufficient 1N H₂ SO₄ to decrease the pH to 4, and sterilizeat 121° C. for 20 minutes.

c) 25 g/l ampicillin addition (300 ml):

Prepare a 25 g/l ampicillin solution by dissolving 9 g of ampicillininto 360 ml of R.O. water. Titrate the suspension to pH 8.0 by adding 1NNaOH in a dropwise manner. Sterilize the resulting solution using a 0.45micron filter, refrigerate, and use within 24 hours.

A 16-liter fermentor containing 9 liters of medium RB6 or RB7(post-sterilization) was inoculated with the 30 ml of broth from a shakeflask. The procedures for storing the culture and for preparing the seedculture are as follows:

B) Ampoule and Seed Preparation Procedures

1) Prepare Petri dishes using the following medium:

    ______________________________________                                        Ingredient      Concentration                                                                             Amount                                            ______________________________________                                        Bacto-agar      15 g/l      3.75 g                                            Trypticase soy-agar                                                                           40 g/l      10.0 g                                            ______________________________________                                    

Q.S. the above ingredients to 250 ml with R.O. water, sterilize theresulting solution in a 500 ml Erlenmeyer flask sealed with a cottonplug at 121° C. for 20 minutes, and then place the flask in a 50° C.water bath. Transfer the culture medium from the heated flask intopresterilized Petri dishes. Allow the dishes to dry overnight under alaminar flow hood with the dish covers propped open. Store the covereddishes in the inverted position.

2) Prepare single colonies of recombinant E. coli by streaking the aboveprepared Petri dishes with the contents of a single ampule of E. colithat was stored under the gaseous phase of liquid nitrogen. Incubate thePetri dishes at 28° C. for 2 days.

3) Prepare 3 shake flasks containing the following medium:

    ______________________________________                                        Ingredient      Concentration                                                                             Amount                                            ______________________________________                                        K.sub.2 HPO.sub.4                                                                             2.6     g/l     0.078  g                                      Na(NH.sub.4)HPO.sub.4.4H.sub.2 O                                                              11.0    g/l     0.33   g                                      Citric Acid.H.sub.2 O                                                                         2.1     g/l     0.063  g                                      (NH.sub.4).sub.2 SO.sub.4                                                                     0.66    g/l     0.020  g                                      MgSO.sub.4.7H.sub.2 O                                                                         0.99    g/l     0.030  g                                      Glycerol        5.0     g/l     0.15   g                                      Yeast Extract   10.0    g/l     0.30   g                                      R.O. Water                      29.0   ml                                     ______________________________________                                    

Seal the above shake flasks with cotton plugs, sterilize them at 121° C.for 20 minutes, and then allow them to cool to room temperature beforeuse.

4) Inoculate each of the above flasks by aseptically transferring asingle colony to it. Place the shake flasks in a controlled environmentincubator shaker set to 320 rpm and 28° C. Sample one of the shakeflasks periodically and determine the absorbance at 550 nm of one ml ofsample diluted with 9 ml of 1% formaldehyde. When an absorbance of 0.3is attained, refrigerate the remaining two shake flasks and use one ofthese flasks as a fermentation seed within 24 hours after refrigeration.

5) Prepare 6 shake flasks containing the following medium:

    ______________________________________                                        Ingredient      Concentration                                                                             Amount                                            ______________________________________                                        K.sub.2 HPO.sub.4                                                                             2.6     g/l     0.052  g                                      Na(NH.sub.4)HPO.sub.4.4H.sub.2 O                                                              11.0    g/l     2.2    g                                      Citric Acid.H.sub.2 O                                                                         2.1     g/l     0.42   g                                      (NH.sub.4).sub.2 SO.sub.4                                                                     0.66    g/l     0.14   g                                      MgSO.sub.4.7H.sub.2 O                                                                         0.99    g/l     0.20   g                                      Glycerol        5.0     g/l     1.0    g                                      Yeast Extract   10.0    g/l     2.0    g                                      R.O. Water                      298.0  ml                                     ______________________________________                                    

Seal the above shake flasks with cotton plugs, sterilize them at 121° C.for 20 minutes, and allow them to cool to room temperature before use.

6) Prepare a 25 g/l ampicillin solution by dissolving 9 grams ofampicillin into 360 ml of R.O. water. To completely dissolve theampicillin, titrate the suspension to pH 8.0 by adding 1N NaOH in adropwise manner. Sterilize the solution using a 0.45μ filter andrefrigerate. Use the solution within 24 hours.

7) Inoculate the above shake flasks by aseptically adding 1 ml of seedand 0.3 ml of the ampicillin solution to each shake flask. Place theshake flasks in a controlled environment incubator shaker set to 320 rpmand 28° C. Sample one of the shake flasks periodically and determine theabsorbance of one ml of sample diluted with 9 ml of 1% formaldehyde at550 nm. When an absorbance of 0.1 is attained, refrigerate the remainingshake flasks.

8) Add 67 g of glycerol to each shake flask prior to filling ampoules.

9) When using a fixed delivery pump to fill ampoules, set the deliveryvolume to 1 ml. Ice and occasionally stir the culture while filling theampoules. (The ampoules must be chilled prior to filling).

The pH of the fermentation broth is controlled using ammonium hydroxide(50 wt %) on/off pH control. The agitation and aeration rates are set to600 rpm and 13.5 slm, respectively, and the backpresure, pH, andtemperature in the fermentor are controlled at 10 psig, 7.2° and 27° C.,respectively.

BST-1 was grown on medium RB6 at the 9 liter scale at 27° C. Dark polarinclusion bodies were formed after spontaneous induction at a celldensity of 0.5 g/l (dry wt.) was exceeded. BSt and cell yields of 1.59g/l and 5.1 g/l, respectively, were obtained after 32 hours offermentation.

EXAMPLE 6

Plasmid Maintenance Stability with pURA-m4

A) Stability of pURA-m4 in BST-1 Shake-Flask Cultures

The maintenance stability of pURA-m4 in strain BST-1 was quantitatedduring experiments conducted with cultures grown at 28° C. for severalgenerations in complex liquid medium without ampicillin. Aliquots weresuccessively obtained from cultures and subcultured at a 10⁻⁵ thdilution into flasks containing fresh complex medium without ampicillin.Following incubation at 28° C., the process was repeated. At thebeginning and end of each passage cycle, culture samples were obtained,diluted, and plated on plates on complex medium, either unsupplementedor supplemented with 40 μg/ml ampicillin, and incubated at 28° C.Quantitation of the total viable population (without ampicillin) allowedestimation of the total number of generations (n) that transpired duringeach passage cycle, according to the following:

    n=(lnX-lnX.sub.o)/ln2

Comparisons of the viable populations quantitated on both media allowedestimation of the plasmid stability frequency (PSF), according to thefollowing:

    PSF=(CFU/ml on Ap plates)/(CFU/ml on unsupplemented plates)

The data showed that plasmid pURA-m4 remained stable throughout anextended period of growth and up to 131.9 generations, as ≧95% of thebacteria were detected as being ampicillin-resistant.

B) Stability of pURA-m4 in Fermentor Cultures of BST-1

The stability of pURA-m4 in strain BST-1 was quantitated duringfermentations at the 5000-liter scale in media without ampicillin.Sample aliquots were aseptically obtained at different times during thecourse of fermentations and handled as described above (Example 6, A) toestimate the number of bacterial generations that had transpired and theplasmid stability frequency. The data showed that pURA-m4 was completelystable throughout the 18.4 generations of the fermentation.

EXAMPLE 7 Expression of Porcine Somatotropin (PSt)

A) Construction of Expression Vector pTrp-conSD

Referring now to Chart 8, to make pTrp-conSD, oligonucleotides are usedto replace the HpaI-HindIII region in pSK4, as shown in Chart 8. pSK4 istreated with HindIII, bacterial alkaline phosphatase (BAP) and HpaI toproduce fragment 16. Fragment 17 is produced synthetically and is adouble-stranded DNA sequence containing four oligonucleotides which areassembled and then purified. Fragments 16 and 17 are ligated togetherand cloned. Clones with restriction patterns characteristic of the Trppromoter are chosen and are designated pTrp-conSD. The sequencedownstream of the Trp promoter in pTrp-conSD is verified by sequencing.Expression vector pTrp-conSD contains the promoter and operator regionof the tryptophan operon, and an E. coli consensus Shine-Dalgarno(conSD, GGAGG) sequence in a pBR322 background with ampicillinresistance.

B) Construction of a Plasmid Containing PSt

Referring now to Chart 9, the method for preparation of the cDNA for PStis known (P. H. Seeburg et al., DNA 2:37-45, 1983; and European Patentapplication, 111,389, U.S. Ser. No. 439,977). The cDNA is cloned intothe PstI site of pBR322 by GC tailing. The fulllength cDNA for PStincluding the 5' and 3' untranslated regions can be excised from thevector by PstI digestion. This PstI fragment (fragment 19, 900 bp)contains two HgiAl sites, one in the presequence and one at the 15thcodon of the mature sequence. The 660 bp HgiAl - PstI fragmentcontaining the truncated PSt cDNA sequence is cloned into the expressionvector pTrp-conSD together with two oligonucleotides. As shown in Chart9, pTrp-conSD is treated with KpnI, Klenow fragment and ClaI to producefragment 18. Fragment 18 is then ligated to fragments 20 and 21.Fragment 20 contains the PSt cDNA truncated for the first 14 codons ofthe mature PSt sequence with a HgiAl sticky end at the 5'-end andblunted PstI at the 3'-end. Fragment 21 is the annealed product of twocomplimentary oligonucleotides which contain the first 14 codons for themature PSt, the translation initiation codon, ATG, a ClaI sticky end forligating into pTrp-conSD, and a HgiAl sticky end for ligating to the PStfragment. In the resulting construct, pTrp-PStl, the expression of PStis under the control of the Trp promoter in the pBR322 background. Thecodon for the fourth alanine in this PSt is changed from GCC in nativecDNA to GCT.

In a similar manner, pTrp-PStc is constructed where no changes in thePSt cDNA are made.

C) Introduction of the PStl Gene Into the pURA Expression Vector

The construction of a PSt expression vector using the chimeric runawayreplicon of the URA vectors and the Trp promoter in combination with aconsensus Shine-Dalgarno sequence is detailed herein.

Referring to Chart 10, the pURA vector is digested with the EcoRI andBamHI restriction enzymes and fragment 22 (4.6 kb) containing the doublereplication ori and the ampicillin resistance gene is isolated aspreviously described.

The pTrp-PStl vector (Chart 9) digested with restriction enzymes EcoRIand BamHI and fragment 23 (900 bp) is isolated as previously described.Fragment 23 contains the trp promoter, the consensus ribosomal bindingsite and the modified porcine somatotropin gene. Fragments 22 and 23 areligated to yield pURA-PStl (Chart 10). Plasmid pURA-PStl is thentransformed into competent cells of E. coli. Confirmation of the cloningis made by restriction digestions of the clones and DNA sequencing.

This vector does not require a transcriptional terminator at the end ofthe porcine gene. The DNA sequence downstream of the coding region ofthe gene which originated from the cDNA has sufficient transcriptionaltermination activity in E. coli.

D) Induction of PSt Expression in E. coli.

Plasmid pURA-PStl is transformed into competent D112 cells and inducedas previously described for rBSt. Procedures for the isolation of rPStare also as described for rBSt. Expression levels have reached 25% oftotal cellular protein.

EXAMPLE 8 Expression of Porcine Somatotropin (PSt) with the TrpLShine-Delgarno Sequence

A) Construction of Expression Vectors pTrp-trpL-PStc and pTrp-trpL-PStl

To further evaluate the expression levels of the porcine geneconstructions PStc and PStl , these genes were placed under control ofthe Trp promoter and the trpL Shine-Delgarno sequence.

Referring to Chart 11, expression vector pTrpl (Example 1) was used asthe source of the Trp promoter and the TrpL Shine-Delgarno sequence.pTrpl is first digested with KpnI and treated with Klenow fragment togenerate a blunt end. The DNA is then cut with ClaI and the large vectorfragment (fragment 24) is purified as described above. A PstI digestionof the cDNA clone of the PSt gene (Example 5) is treated with Klenowfragment to generate a blunt end and is then cut with HgiA1. Thisfragment (fragment 20) is then purified. A synthetic double-stranded DNAlinker fragment is constructed from two DNA oligonucleotides. Twosynthetic linker fragments are made. The first contains the change fromGCC to GCT for the second alanine codon of PSt (fragment 25) and thesecond linker contains unmodified PSt cDNA sequence (fragment 26). Thefragments 20, 24 and 25 are ligated to produce pTrp-trpL-PSt1, and thefragments 20, 24 and 26 are ligated to produce pTrp-trpL-PStc.

B) Construction of pURA Vectors Which Contain Either the trpL-PStc orthe trpL-PStl Gene

The construction of PSt expression vectors using the chimeric runawayreplicon of the pURA vectors and the trp promoter in combination withthe trp leader Shine-Delgarno sequence (trpL) is detailed herein (Chart12).

The pURA vector is digested with EcoRI and BamHI restriction enzymes andfragment 22 (4.6 kb) is isolated. The pTrp-trpL-PStc and pTrp-trpL-PStlvectors (Chart 11) are digested with restriction enzymes EcoRI and BamHIand a fragment of approximately 900 bp is isolated from each digestion.The fragments so produced from pTrp-trpL-PStc and from pTrp-trpL-PStlare designated fragments 27 and 28, respectively. Fragments 22 and 27are ligated to form pURA-trpL-PStc. Fragments 22 and 28 are ligated toform pURA-trpL-PStl . Confirmation of the clonings is made byrestriction analysis and DNA sequencing.

C) Induction of pURA-trpL-PStc and pURA-trpL-PStl

Plasmids pURA-trpL-PStc and pURA-trpL-PStl are transformed intocompetent cells of strain D112 and induced as previously described forrBSt. Samples of the inductions are analyzed by SDS-PAGE, Coomassie Bluestaining, and gel scanning. Porcine somatotropin was not detectable withthe pURA-trpL-PStc induction. The induction of pURA-trpL-PStl showedporcine somatotropin being expressed at a level of about 5% of the totalvisible protein.

EXAMPLE 9 Expression of Human Interleukin-1β (IL-1β)

A) Construction of a Plasmid Containing the IL-1β Gene

The cloning of the human IL-1β sequence originated from a cDNA sequencecontaining part of the 5' untranslated region and all of the codingregion of IL-1β and ending after the stop codon. This cDNA sequence wasconstructed from a human SK-Hep-1 cell line and cloned into pTrpl(Chart 1) to yield pTrp-ILl -1. Another subcloning was done to constructpTrp-conSD-HC-ILl -1, containing the mature IL-1β sequence. Theconstruction of these plasmids has been published (D. B. Carter, et al.,1988, Proteins: Structure, Function and Genetics, in press, which isincorporated herein by reference). Plasmid pTrp-conSD-HC-ILl -1 is shownin Chart 14.

Plasmid pTrp-conSD-HC-ILl -1 was digested with EcoRI and BamHI and theresulting 790 base-pair fragment was isolated and then mixed and ligatedwith the 4.6 kilobase-pair EcoRI/BamHI fragment of pURA (Chart 6) toproduce pURA-IL-1-1A. pURA-IL-1-1A was subsequently digested with BamHIand mixed and ligated with the BamHI fragment 14 (Chart 7) of pVV202T toyielded pURA-ILl -1C.

B) Spontaneous Induction of IL-1β Expression

The vector pURA-ILl -1C was transformed into competent cells of BST-1C.Seed cultures of BST-1C (pURA-ILl -1C) were grown in shake flaskscontaining defined inorganic salts medium supplemented with glucose,yeast extract, and tryptophan, and incubated at 28° C. throughout.Culture aliquots were subcultured into defined inorganic salts mediumsupplemented with glucose and casamino acids, and the cultures were thenincubated at 28° C. throughout. Culture samples were periodicallywithdrawn and subjected to SDS-PAGE analyses, followed by densitometricscanning of the gels, to quantitate the level of IL-1β produced. Thedata in Table 5 show that IL-1β production was spontaneously inducedunder such conditions, and that such spontaneous induction resulted in alevel of IL-1β which was very similar to that which was attained by thesame culture in identical medium, but which was shifted from 28° C. to37° C. at a culture O.D. 1 A₅₅₀ (thermally induced).

                                      TABLE 1                                     __________________________________________________________________________    Bovine somatotropin - cDNA sequence and corresponding amino acid              __________________________________________________________________________    sequence.                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                    __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Synthetic Oligonucleotides Used to Complete the Synthetic 5' End of           __________________________________________________________________________    rBSt                                                                          Oligonucleotide 1. Native cDNA for pTrpBSt102                                  ##STR12##                                                                     ##STR13##                                                                    Oligonucleotide 2. pTrpBStm4                                                   ##STR14##                                                                     ##STR15##                                                                    Oligonucleotide 3. pTrpBStm5                                                   ##STR16##                                                                     ##STR17##                                                                    Oligonucleotide 4. pMBSt4                                                      ##STR18##                                                                     ##STR19##                                                                    Oligonucleotide 5. pMBSt12                                                     ##STR20##                                                                     ##STR21##                                                                    Oligonucleotide 6. pTrpBSt[lys]phe                                             ##STR22##                                                                     ##STR23##                                                                    Oligonucleotide 6. pTrpBSt[phe]phe                                             ##STR24##                                                                     ##STR25##                                                                    Oligonucleotide 7. pTrpBStm3                                                   ##STR26##                                                                     ##STR27##                                                                    __________________________________________________________________________

                  TABLE 3                                                         ______________________________________                                        Spontaneous Induction of Runaway Plasmid Replication                          and rBSt Synthesis                                                            Time Post-  rBSt       Relative Plasmid                                       Inoculation Titer      Content                                                (hours)     (g rBSt/liter)                                                                           (per 10.sup.10 bacteria).sup.a                         ______________________________________                                         9.0        0.0000       182,741                                              16.0        0.0113       482,268                                              18.0        0.0770       834,673                                              19.0        0.1303     1,063,058                                              22.0        0.4627     1,145,452                                              25.0        0.7396     1,088,053                                              27.0        0.9543     1,152,051                                              29.0        1.0871     1,205,500                                              31.0        1.0975     1,122,224                                              ______________________________________                                         .sup.a Peak area from densitometric trace of covalentlyclosed circular        monomer plasmid band.                                                    

                  TABLE 4                                                         ______________________________________                                        Spontaneous Induction of Runaway Plasmid                                      Replication in the Absence of rBSt Synthesis                                                Time Post-                                                                              Relative Plasmid                                                    Inoculation                                                                             Content                                               Plasmid       (hours)   (per 10.sup.10 bacteria).sup.a                        ______________________________________                                        pURA--m4      16.0      591,763                                               pURA--m4      22.0      1,249,226                                             pURA--m4      31.0      1,149,770                                             pURA4 Δbgh.sub.E/H                                                                    18.0      540,450                                               pURA4 Δbgh.sub.E/H                                                                    24.0      970,199                                               pURA4 Δbgh.sub.E/H                                                                    30.0      1,755,525                                             ______________________________________                                         .sup.a Peak area from densitometric trace of covalentlyclosed circular        monomer plasmid band.                                                    

                  TABLE 5                                                         ______________________________________                                        Spontaneous Induction of IL--1β                                          Production with the Runaway Vector pURA--IL1--1C                              Culture       Time Post-                                                                              IL13 1β                                          Incubation    Inoculation                                                                             Produced                                              Temperature   (hours)   (% total protein)                                     ______________________________________                                        28° C.  0.0      3.9                                                                  3.0      2.3                                                                 10.0      3.5                                                                 28.0      18.8                                                  28° C. → 37° C.                                                        28.0      20.6                                                  ______________________________________                                    

CHART 1 Construction of pTrpl

(a) pSK4 is cut with ClaI and BamHI to isolate fragment 1 (4.2 kb).##STR28##

(b) The synthetic sequence of double-stranded DNA, fragment 2, isligated to fragment 1 to yield pTrpl (4.3 kb). ##STR29##

CHART 2 Construction of pTrp-BStm1

(a) pLG23 is cut with PvuII to isolate fragment 3 (494 bp). ##STR30##

(b) pTrpl is treated with KpnI and Klenow to yield fragment 4 (4.3 kb).##STR31##

(c) Fragment 3 is ligated to fragment 4 to yield pTrp-BStm1. kb)##STR32##

CHART 3 pTrp-BStm1b

(a) pTrp-BStm1 is cut with MstII and BamHI to isolate fragment 5 (4.8kb). ##STR33##

(b) Four oligonucleotides are assembled to yield fragment 6. ##STR34##

(c) Fragment 6 is ligated to fragment 5 to yield pTrp-BStm1b ##STR35##

CHART 4 pTrp-BStm4

(a) pTrp-BStm1b is cut with MstII and ClaI to isolate fragment 7 (4.3kb). ##STR36##

(b) pTrp-BStm1b is cut with PvuII and MstII to isolate fragment 8 (450bp). ##STR37##

(c) Four oligonucleotides are assembled to yield fragment 9.Oligonucleotide 2 (Table 2) ##STR38##

(d) Fragments 7, 8 and 9 are ligated to yield pTrp-BStm4 (4.8 kb).##STR39##

CHART 5 Construction of p50-BStm4

(a) Plasmid pBEU50 was digested with EcoRI and BamHI and the large 10 kbfragment 10 is gel isolated. ##STR40##

(b) Plasmid pTrp-BStm4 (Chart 4) was digested with EcoRI and BamHI toyield fragment 11 (0.872 kb) which is gel isolated. ##STR41##

(c) Fragments 10 and 11 are annealed and ligated using T4 ligase toobtain p50-BStm4. ##STR42##

CHART 6 Construction of pURA

(a) Vector pBEU-17 (13.8 kb) was digested with AhaII and NdeI to yieldfragment 12 (2.4 kb). ##STR43##

(b) Fragment 12 is inserted and ligated into pBR322 (4.4 kb) which waspreviously digested with NarI and NdeI to yield pURA (5.0 kb). ##STR44##

CHART 7 Construction of pURA-m4

(a) p50-BStm4 (Chart 5) is digested with EcoRI and BamHI to yieldfragment 13 (875 bp) containing the Trp promoter and rBSt gene.##STR45##

(b) pVV202T is digested with BamHI to yield fragment 14 (350 bp)containing a transcription terminator. ##STR46##

(c) pURA (Chart 6) is digested with EcoRI and BamHI to yield fragment 15(4.6 kb) and ligated with fragments 13 and 14 to yield pURA-m4 (5.8 kb).##STR47##

CHART 8 Construction of pTrp-conSD

(a) pSK4 is treated with HindIII, BAP and HpaI to obtain fragment 16(4.6 kb). ##STR48##

(b) The synthetic sequence of double-stranded DNA fragment 17 is ligatedto fragment 18 to yield pTrp-conSD (4.6 kb). ##STR49##

CHART 9 pTrp-PSt1

(a) pTrp-conSD is treated with KpnI, Klenow and ClaI to obtain fragment18 (4.6 kb). ##STR50##

(b) A PstI fragment (900 bp) containing the full length cDNA of PSt isisolated (Fragment 19). Fragment 19 is treated with Klenow and HgiA1 toisolate fragment 20 (660 bp). ##STR51##

(c) The synthetic sequence of double-stranded DNA fragment 21 is ligatedto fragments 19 and 20 to yield pTrp-PSt1. ##STR52##

CHART 10 Construction of pURA/PSt1

(a) Plasmid pURA is digested with EcoRI and BamHI and fragment 22 isisolated. ##STR53##

(b) Plasmid pTrp-PStl is cut with EcoRI and BamHI and fragment 23 (900bp) is isolated. Fragments 22 and 23 are ligated to form pURA/PStl.##STR54##

CHART 11 pTrp-trpL-PStc and pTrp-trpL-PStl

a) pTrpl (Chart 1) is treated with KpnI, Klenow and ClaI to obtainfragment 24 (4.6 kb). ##STR55##

b) A PstI fragment (900 bp) containing the full length cDNA of PSt isisolated (Fragment 19). Fragment 19 is treated with Klenow and HgiA1 togenerate fragment 20 (660 bp). ##STR56##

c) The synthetic sequence of double-stranded DNA fragment 25 is ligatedto fragments 24 and 20 to yield pTrp-trpL-PStl and the syntheticfragment of double-stranded DNA fragment 26 is ligated to fragments 24and 20 to yield pTrp-trpL-PStc. ##STR57##

CHART 12 Construction of pURA-trpL-PStc and pURA-trpL-PStl

a) Plasmid pURA is digested with EcoRI and BamHI and fragment 22 (Chart10) is isolated. ##STR58##

b) Plasmids pTrp-trpL-PStc and pTrp-trpL-PStl are cut with EcoRI andBamHI and fragments 27 and 28 (900 bp) are isolated. Fragments 22 and 27are ligated to form pURA-trpL-PStc. Fragments 22 and 28 are ligated toform pURA-trpL-PStl. Both vectors which differ by a single base pair canbe represented as shown below. ##STR59##

CHART 13 Deletion of the pURA-m1 Gene

(a) Plasmid pURA-M4 (Chart 7) was digested with EcoRI and HindIII andthe overhanging 5'-ends were blunted by filling with Klenow fragment anddNTP's to generate fragment 29. ##STR60##

(b) Fragment 29 was ligated to yield pURA4 Δbgh_(E/H). ##STR61##

CHART 14 Plasmid pTrp-conSD-HC-ILl-1 ##STR62##

We claim:
 1. An expression plasmid capable of runaway at a constanttemperature below 34° C. which is useful for transforming a host celland permitting the host cell to produce a heterologous protein, whichplasmid comprises a mutant R1 replicon from pBEU-17, an orgin ofreplication from pBR322, and a cDNA encoding the heterologous protein.2. A plasmid according to claim 1, wherein the host cell is E. coli andthe cDNA encodes a bovine or porcine somatotropin.
 3. A plasmidaccording to claim 2, wherein the first four codons of the cDNA areselected from the group consisting of:
 4. A plasmid according to claim 3wherein the first four codons of the cDNA are GCC TTC CCA GCT.
 5. An E.coli host cell comprising a mutation of the rpoH112 allele or hflB29allele, and a plasmid of claim
 1. 6. A host cell according to claim 5wherein the mutation is the rpoH112 allele.
 7. A host cell according toclaim 5 wherein the mutation is the hflB29 allele.
 8. An E. coli hostcell comprising a mutation of the rpoH112 allele or hflB29 allele, and aplasmid of claim
 3. 9. A host cell according to claim 8 wherein themutation is the rpoH112 allele.
 10. A host cell according to claim 8wherein the mutation is the hflB29 allele.
 11. A host cell according toclaim 8 wherein the mutation is the hflB29 allele and the first fourcodons of the cDNA are GCC TTC CCA GCT.
 12. A host cell according toclaim 8 wherein the plasmid encodes porcine somatotropin.
 13. A methodof expressing a heterologous gene comprising culturing a host cellaccording to claim
 5. 14. A method of expressing a heterologous genecomprising culturing a host cell according to claim
 8. 15. E. coli hostcell BST-1C (NRRL B-18303) or D112 (NRRL B-18168).
 16. The E. coli hostcell according to claim 15 which is BST-1C (NRRL B-18303).